POS0252 MYOFIBROBLASTS MAINTAIN Th1 and Tc1 POLARIZATIONS IN GIANT CELL ARTERITIS
Authors: H. Greigert, A. Ramon, C. Gerard, M. Ciudad, C. Cladiere, C. Genet, L. Arnould, C. Creuzot-Garcher, L. Martin, G. Tarris, S. Audia, M. C. Cid, B. Bonnotte, M. Samson
Journal: Annals of the Rheumatic Diseases (2022)

Background Giant cell arteritis (GCA) is a large-vessel vasculitis mainly involving the aorta and cranial arteries. It is the most frequent vasculitis in adults over 50 years. When they are stimulated by interferon-gamma (IFN-γ), vascular smooth muscle cells (VSMC) contribute to GCA pathogenesis by producing chemokines triggering the recruitment of pro-inflammatory T cells and monocytes (1). Objectives Current knowledge about the interaction between resident cells of the vascular wall (VSMC, myofibroblasts [MF]) and immune cells is limited. The aim of our research was to better characterize the interactions between VSMC, MF and T cells in GCA. Methods Fresh fragments of temporal artery biopsies (TAB) performed at Dijon university hospital (France) were prospectively sent to our research unit. Fresh sections of positive and negative TAB were fixed and embedded in optimal cutting temperature OCT and stored at -80°C. Then, cryostat sections were fixed, permeabilized, blocked and incubated with primary antibodies (anti-alpha smooth muscle actin [α-SMA], anti-myosin heavy chain 11 [MHC11], anti-Desmin, anti CD90, anti-CD45, anti-HLA-DR, anti-phospho STAT1 [pSTAT1] and anti-pSTAT3) and secondary antibodies for confocal microscopy analyses. Fresh sections of healthy TAB were embedded in MATRIGEL and covered by DMEM to obtain vascular cells in culture. Cells were treated with trypsina-EDTA between each passage. Vascular cells were used after 4-7 doubling passages. Cells were analyzed by immunofluorescence, flow cytometry and RT-PCR and their proliferation was evaluated by impedancemetry (iCELLigence system). Peripheral blood mononuclear cells (PBMC) and vascular cells thus obtained were co-cultured for 7 days in different conditions. Vascular cells were cultured in the presence or absence of IFN-γ and tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) and soluble receptor of IL-6 for 72 hours. When cells reached confluence, they were cultured alone or with allogenic PBMC activated with anti-CD3/CD28 microbeads. After 7 days of culture, cells were separated with a treatment with EDTA and studied by flow cytometry. Results Confocal microscopy analyses of GCA arteries showed that neointima was mainly composed of myofibroblasts (MF) (α-SMA + Desmin + MHC11 low CD90 + ) in contact with CD45 + cells and that MF expressed HLA-DR, the phosphorylated form of STAT1 (pSTAT1) and in a lesser extent pSTAT3, strongly suggesting the activation of the IFN-γ signaling pathway rather than the IL-6 pathway. The phenotype of cultured vascular cells isolated from fresh TAB was consistent with MF. When MF were exposed to IFN-γ and TNF-α in vitro , their proliferation capacity decreased and their levels of expression of HLA-DR and CD86 increased (median fluorescence intensity [MFI] from 0 to 57 [p=0.03] and from 34 to 103 [p=0.03], respectively). In addition, co-cultures of MF and activated PBMC revealed that MF maintained the polarization of T cells into Th1 and Tc1 cells (p≤0.001) and to a lesser extent into Th17 and Tc17 cells (p=0.03). This effect was even more significant when MF were previously exposed to IFN-γ and TNF-α but not when they were exposed to IL-6. Conclusion Our results show that myofibroblasts are present in the neointima of GCA patients and that these MF activate signaling pathways indicative of IFN-γ exposure. Moreover, these MF, especially when exposed to IFN-γ, maintain the polarization of T cells into Th1 and Tc1 cells, which contributes to amplify the production of IFN-γ and thus initiate a pro-inflammatory amplification loop that likely participates in vascular inflammation and remodelling. References [1]Corbera-Bellalta M, Planas-Rigol E, Lozano E, Terrades-Garcia N, Alba MA, Prieto-Gonzalez S, et al. Blocking interferon gamma reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis. Ann Rheum Dis 2016;75:1177-86. Disclosure of Interests None declared